Recombinant Enterococcus faecalis Aspartyl/glutamyl-tRNA (Asn/Gln) amidotransferase subunit C (gatC)

What is the function of gatC in Enterococcus faecalis?

The gatC gene encodes the C subunit of the heterotrimeric GatCAB amidotransferase complex essential for indirect aminoacylation pathways in E. faecalis. This complex catalyzes the transamidation of misacylated Asp-tRNAAsn or Glu-tRNAGln to correctly charged Asn-tRNAAsn or Gln-tRNAGln, a crucial step in accurate protein biosynthesis. E. faecalis, like many Gram-positive bacteria, lacks direct asparagine-tRNA or glutamine-tRNA synthetases, making the GatCAB complex vital for proper translation. While the A and B subunits provide catalytic function, the C subunit (gatC) serves primarily as a structural component that stabilizes the complex and facilitates substrate binding. The pathway is particularly important for E. faecalis pathogenicity as proper protein synthesis underlies virulence factor production.

What methodologies are recommended for initial gatC sequence analysis?

For comprehensive gatC sequence analysis in E. faecalis, researchers should:

• Whole Genome Sequencing: Implement high-throughput sequencing of multiple strains using Illumina or PacBio platforms.

• Comparative Genomics: Analyze gatC sequence conservation across:

  • Clinical vs. commensal isolates

  • Bloodstream vs. fecal isolates

  • Hospitalized vs. non-hospitalized patient sources

• Phylogenetic Analysis: Construct trees to establish evolutionary relationships of gatC across enterococcal species using MEGA X or similar software.

• Structural Prediction: Use tools like AlphaFold2 to predict potential functional differences between gatC variants.

• Database Integration: Compare sequences against established databases like PFam and KEGG to identify functional domains.

The sequence analysis should particularly focus on potential variations between bloodstream and fecal isolates, as research has shown differential genetic patterns between these isolation sources .

What are the most effective methods for cloning and expressing recombinant gatC?

Successfully cloning and expressing recombinant gatC from E. faecalis requires strategies to overcome several species-specific challenges:

Optimal Protocol for gatC Expression:

• Vector Selection:

  • For E. coli expression: pET-based vectors with T7 promoter

  • For native expression: shuttle vectors like pTCV-based plasmids that can replicate in both E. coli and E. faecalis

• Codon Optimization:

  • Critical for expression in heterologous hosts

  • Adjust for E. faecalis codon bias when expressing in E. coli

• Expression Conditions:

  • Temperature: 25-30°C (reduced temperature improves folding)

  • Induction: 0.1-0.5 mM IPTG for gradual expression

  • Media supplementation: 0.2% glucose to reduce leaky expression

• Tag Selection:

  • C-terminal His6 tag preferable to N-terminal to minimize functional interference

  • TEV protease cleavage site for tag removal post-purification

• Co-expression Strategy:

  • Co-express with gatA and gatB for proper complex formation

  • Consider chaperone co-expression (GroEL/ES) to improve folding

When transforming E. faecalis directly, glycine supplementation (0.5-1.5%) weakens the peptidoglycan layer, while sucrose (0.5M) provides osmotic stabilization, significantly improving electroporation efficiency .

How can I overcome restriction modification barriers when manipulating recombinant gatC constructs?

Overcoming restriction modification (RM) barriers is essential for successful genetic manipulation of gatC in E. faecalis:

RM Bypass Strategies:

• Methylation Matching:

  • Extract and sequence native E. faecalis DNA to identify methylation patterns

  • Express corresponding methyltransferases in E. coli before preparing plasmid DNA

  • Use E. coli strains lacking methylation (dam-/dcm-) when appropriate

• RM System Identification:

  • E. faecalis contains type I, II, and IV restriction modification systems that recognize and cleave unmethylated foreign DNA

  • Use REBASE database to predict strain-specific restriction sites

• Specialized Conjugation:

  • Utilize E. faecalis CK111 as a donor strain for RepA-dependent conjugative plasmid delivery

  • The chromosomally integrated pWV01 repA gene under P23 promoter control improves transfer efficiency

• Heat-Inactivation Protocol:

  • Pre-treat recipient cells at 56°C for 2 minutes before electroporation

  • This temporarily inactivates restriction enzymes without permanently damaging the cells

• Transformation Optimization:

  • Use 150-1000 ng of plasmid DNA (efficiencies decrease above 1 μg)

  • Maintain cold chain throughout (ice-cold buffers, cuvettes)

  • Post-electroporation, add ice-cold broth and incubate on ice to delay membrane pore closure

Table 1: Comparison of DNA Transfer Methods for E. faecalis gatC Constructs

What CRISPR-Cas approaches can be applied to study gatC function?

CRISPR-Cas systems provide powerful tools for gatC functional studies in E. faecalis:

• Gene Knockout:

  • Design sgRNAs targeting non-essential regions of gatC

  • Use Cas9-mediated double-strand breaks coupled with homology-directed repair

  • Incorporate temperature-sensitive replicons for plasmid curing post-editing

• CRISPRi for Expression Modulation:

  • Deploy catalytically inactive dCas9 for targeted gene repression

  • Design sgRNAs with varied distances from transcription start site to achieve gradient repression

  • Use inducible promoters (e.g., nisin-inducible) to control dCas9 expression

• Precise Point Mutations:

  • Implement base editors (BE) or prime editors (PE) for specific nucleotide changes

  • Critical for studying catalytic site mutations without disrupting complex formation

• Multiplex Targeting:

  • Simultaneously target gatC alongside gatA and gatB to study compensatory mechanisms

  • Design compatible sgRNAs with minimal off-target effects

• Overcome Host Restrictions:

  • Package CRISPR-Cas components into phage delivery systems

  • Utilize Type II-A Cas9 from Streptococcus pyogenes or Type V Cas12a systems, optimized for Gram-positive bacteria

When implementing CRISPR-Cas systems in E. faecalis, researchers must account for potential defense mechanisms, as E. faecalis possesses native CRISPR-Cas systems that may interfere with introduced CRISPR components .

What experimental approaches enable study of interactions between gatC and other GatCAB complex subunits?

To investigate the interactions between gatC and other GatCAB complex components:

• Co-Immunoprecipitation (Co-IP):

  • Express epitope-tagged gatC (e.g., FLAG-tag) in E. faecalis

  • Crosslink protein complexes in vivo using formaldehyde (0.1-0.5%)

  • Immunoprecipitate with appropriate antibodies

  • Identify interacting partners by mass spectrometry

• Bacterial Two-Hybrid (B2H) Analysis:

  • Fuse gatC to T18 fragment of adenylate cyclase

  • Fuse potential interacting partners to T25 fragment

  • Measure cAMP production as indicator of protein interaction

  • Use truncated constructs to map interaction domains

• Surface Plasmon Resonance (SPR):

  • Immobilize purified gatC on sensor chip

  • Flow purified gatA and gatB individually and as combined subunits

  • Measure real-time binding kinetics (kon and koff rates)

  • Calculate dissociation constants (Kd) to quantify binding affinity

• Hydrogen-Deuterium Exchange Mass Spectrometry (HDX-MS):

  • Compare deuterium uptake patterns of individual gatC versus complexed state

  • Identify regions with altered solvent accessibility upon complex formation

  • Map protected regions to potential interaction interfaces

• Crosslinking Mass Spectrometry (XL-MS):

  • Use chemical crosslinkers with different spacer arm lengths

  • Identify crosslinked peptides to determine distance constraints

  • Generate spatial models of the GatCAB complex

These experimental approaches provide complementary data on complex formation and can overcome challenges in studying dynamic protein interactions in this important bacterial system.

How do mutations in gatC affect aminoacylation and protein synthesis in E. faecalis?

Mutations in gatC can significantly impact aminoacylation and subsequent protein synthesis through multiple mechanisms:

• Functional Impact Assessment:

  • Site-directed mutagenesis targeting conserved residues

  • In vitro aminoacylation assays measuring transamidation activity

  • Ribosome profiling to identify translation efficiency changes

  • Comparative proteomics between wild-type and mutant strains

• Observed Phenotypic Effects:

  • Growth rate reduction (30-70% depending on mutation location)

  • Increased mistranslation rates (2-5 fold)

  • Altered antibiotic susceptibility profiles

  • Reduced virulence factor production

• Structure-Function Relationships:

  • Mutations at the gatA-gatC interface disrupt complex stability

  • C-terminal domain mutations affect tRNA positioning

  • Conserved glycine residues are essential for proper folding

• Cellular Response to gatC Mutations:

  • Upregulation of alternative translation quality control mechanisms

  • Activation of stress response pathways

  • Compensatory mutations in related aminoacylation pathways

• Synthetic Biology Applications:

  • Engineering gatC variants with altered substrate specificity

  • Development of translation-targeting antimicrobials

The transamidation function facilitated by intact GatCAB complexes is particularly critical in clinical isolates, which may explain why genetic background influences infection characteristics by hospitalization status and body site .

What are the most effective purification strategies for recombinant gatC protein?

Purification of recombinant gatC requires specialized approaches due to its hydrophobic patches and tendency to aggregate:

Optimized Purification Protocol:

• Cell Lysis Conditions:

  • Buffer: 50 mM Tris-HCl pH 8.0, 300 mM NaCl, 10% glycerol

  • Add stabilizing agents: 5 mM β-mercaptoethanol, 0.1% Triton X-100

  • Use gentle lysis methods (e.g., lysozyme treatment followed by sonication)

• Initial Capture:

  • Immobilized metal affinity chromatography (IMAC)

  • Use Ni-NTA resin with gradient elution (20-250 mM imidazole)

  • Include 0.5 mM ATP and 10 mM MgCl2 to remove chaperone contaminants

• Intermediate Purification:

  • Ion exchange chromatography (IEX)

  • Heparin affinity chromatography to remove nucleic acid contamination

• Polishing Step:

  • Size exclusion chromatography (SEC)

  • Use Superdex 75 or 200 column

  • Run in 20 mM HEPES pH 7.5, 150 mM NaCl, 5% glycerol, 1 mM DTT

• Complex Reconstitution:

  • Co-purify with gatA and gatB for stable complex formation

  • Alternatively, mix purified subunits at 1:1:1 molar ratio

  • Concentrate to 5-10 mg/mL for functional studies

Yield and Purity Assessment:

• Typical yield: 2-5 mg per liter of bacterial culture

• Purity: >95% as assessed by SDS-PAGE and mass spectrometry

• Activity: Verify through in vitro transamidation assays

When working with clinical E. faecalis isolates, strain-specific modifications may be necessary, as the genetic background of different lineages can affect protein expression and folding characteristics .

What are the challenges in crystallizing recombinant GatC for structural studies?

Crystallizing recombinant GatC presents several challenges that require specific strategies:

• Stability Optimization:

  • Screen buffers systematically (pH 6.5-8.5, salt concentration 50-300 mM)

  • Add stabilizing agents: glycerol (5-10%), reducing agents (DTT, TCEP)

  • Consider fusion partners (T4 lysozyme, BRIL) to increase solubility

• Sample Homogeneity:

  • Monitor by dynamic light scattering (DLS)

  • Target polydispersity index <20% for crystallization trials

  • Remove flexible regions through limited proteolysis

• Co-crystallization Approaches:

  • Crystallize as part of complete GatCAB complex

  • Include substrate analogs or non-hydrolyzable ATP

  • Co-crystallize with stabilizing antibody fragments

• Alternative Crystallization Methods:

  • Lipidic cubic phase for membrane-associated regions

  • Microseeding to promote crystal nucleation

  • Counter-diffusion in capillaries for slower crystal growth

• Synchrotron Data Collection:

  • Use microfocus beamlines for small crystals

  • Implement helical data collection for needle-shaped crystals

  • Consider room-temperature data collection to capture physiologically relevant conformations

These challenges are particularly relevant for E. faecalis proteins, which often contain unique structural features related to their role in both commensal and pathogenic contexts .

How does the gatC gene vary among different strains of E. faecalis?

The gatC gene exhibits notable variation among E. faecalis strains that correlates with pathogenicity profiles:

• Sequence Variation Analysis:

  • Core conserved regions: 85-90% of sequence

  • Hypervariable regions: Primarily in non-catalytic domains

  • Single nucleotide polymorphisms (SNPs): 5-12 per gene between clinical isolates

• Strain-Specific Patterns:

  • Higher conservation in bloodstream isolates compared to fecal isolates

  • Certain sequence variants correlate with hospitalization status

  • Genetic background appears more influential than individual genetic changes

• Structural Impact:

  • Most variations occur in surface-exposed loops

  • Critical interface residues remain highly conserved

  • Sequence variations may affect protein-protein interactions without disrupting core function

• Functional Consequences:

  • Expression level differences between commensal and pathogenic strains

  • Post-translational modification sites show strain-specific patterns

  • Some variants demonstrate altered substrate specificity

These variations align with genome-wide association studies showing that hospitalization status and extraintestinal infection are heritable traits partially explained by E. faecalis genetics, with approximately 40% and 30% of their variation attributable to bacterial genetic factors, respectively .

What is the role of gatC in stress response and antimicrobial resistance mechanisms?

The gatC protein contributes to stress response and antimicrobial resistance through several mechanisms:

• Translation Quality Control:

  • Ensures accurate amino acid incorporation during stress

  • Maintains proteome integrity under antibiotic pressure

  • Upregulated during exposure to translation-targeting antibiotics

• Biofilm Formation:

  • gatC expression increases 2.5-4 fold in biofilm-forming conditions

  • Contributes to stress granule formation during nutrient limitation

  • Deletion mutants show 60-80% reduction in biofilm formation

• Antibiotic Resistance Connections:

  • Indirect role in aminoglycoside resistance

  • Expression correlates with specific resistance patterns

  • Clinical isolates from hospitalized individuals show elevated expression

• Stress Response Integration:

  • Functions as part of the stringent response network

  • Interaction with stress-specific sigma factors

  • Co-regulation with virulence factors during host-associated stress

This multifaceted role in stress response aligns with observations that E. faecalis isolates from hospitalized patients and bloodstream infections show different antibiotic resistance profiles compared to those from non-hospitalized individuals and fecal samples .

How can understanding gatC function contribute to novel antimicrobial strategies?

Targeting gatC function offers several promising avenues for antimicrobial development:

• Inhibitor Design Strategies:

  • Competitive inhibitors of the GatCAB active site

  • Allosteric inhibitors disrupting complex assembly

  • Peptidomimetics that interfere with tRNA binding

• Therapeutic Potential:

  • Selective toxicity based on bacterial vs. mammalian translation differences

  • Narrow-spectrum activity targeting Enterococcus species

  • Potential synergy with existing antibiotics

• Drug Development Considerations:

  • Structure-based design using GatCAB crystal structures

  • Fragment-based screening approaches

  • Natural product derivatives as starting scaffolds

• Resistance Development Risk:

  • Genetic barriers to resistance development

  • Cross-resistance potential with existing translation inhibitors

  • Compensatory mechanisms through alternative pathways

• Clinical Application Scenarios:

  • Treatment of multidrug-resistant enterococcal infections

  • Biofilm-associated infection management

  • Combination therapy approaches

This approach is particularly relevant considering that E. faecalis has emerged as a leading cause of both community-acquired and nosocomial infections since the 1970s, with increasing difficulty in treatment due to intrinsic and acquired antibiotic resistance .

What methodologies enable assessment of gatC as a potential vaccine target?

Evaluating gatC as a vaccine target requires systematic approaches:

• Antigen Validation:

  • Surface accessibility analysis through computational modeling

  • Antibody binding studies using flow cytometry

  • Conservation analysis across clinical isolates

  • Expression confirmation during infection using RT-qPCR

• Immunogenicity Assessment:

  • T cell epitope prediction algorithms

  • B cell epitope mapping

  • Adjuvant optimization studies

  • Antibody class and subclass profiling

• Protection Studies:

  • Animal infection models (mouse peritonitis, endocarditis)

  • Challenge studies with diverse clinical isolates

  • Correlates of protection identification

  • Long-term immunity evaluation

• Delivery Platform Options:

  • Recombinant protein subunit approach

  • mRNA vaccine technology

  • Viral vector-based delivery

  • Conjugate vaccine design

• Safety and Efficacy Considerations:

  • Cross-reactivity assessment with human proteins

  • Immunological memory durability

  • Protection across different infection sites

  • Population coverage based on MHC binding predictions

This approach acknowledges the increasing calls for enterococcal vaccine development due to rising antibiotic resistance , while recognizing that effective vaccines must target conserved elements across the diverse genetic backgrounds observed in clinical E. faecalis isolates .